Non-peptide hormones are involved in coordination of multiple events related to development, differentiation and physiological response to a wide range of stimuli. These hormones bind to intracellular nuclear hormone receptors that mediate the hormonal effect. Nuclear hormone receptors (NHRs) have been identified as ligand-dependent transcription factors that initiate nuclear responses to steroids, retinoids, 1,25 dihydroxyvitamin D.sub.3 and thyroid hormones. Peroxisome proliferator-activated receptors (PPARs) are also members of the NHR family. Based on shared characteristics, a superfamily of nuclear hormone receptors has been recognized. The superfamily also includes structurally related proteins for which no ligand has been identified ("orphan receptors").
Members of the NHR superfamily share structural similarity. The majority exhibit three prinicipal domains: (1) a variable, amino-terminal domain that often is related to transactivating activity; (2) a highly conserved DNA binding domain; and (3) a moderately conserved carboxy-terminal ligand-binding domain. The DNA binding domain has two "zinc finger" motifs, and differences in the sequence of these motifs have been associated with differences in DNA binding or receptor dimerization or its absence. Further, the region immediately carboxy-terminal of the zinc fingers has been implicated in DNA recognition. This region contains two adjacent clusters of amino acids referred to as the "A- and T-boxes." Differences in these regions correlate with the subdivision of the superfamily into four groups: (I) receptors that act as homodimers; (II) receptors that act as both homodimers and heterodimers; (III) receptors that act as monomers; and (IV) receptors that bind exclusively as heterodimers.
NHRs (also termed "nuclear receptors") bind to DNA sequences, called "response elements", in the promoter region of target genes. These response elements have distinct sequence motifs, and the relative orientation and spacing of these sequence motifs are important for receptor binding specificity.
One family of NHRs, the retinoid-related receptors, has been subdivided into a number of classes, including RAR (retinoic acid receptors), RXR (retinoid X receptors), RZR (retinoid Z receptors) and ROR (RAR-related orphan receptors). RZRs were reported to bind to natural retinoid response elements (hexameric (A/G)GGTCA core half-site motif) as monomers (C. Carlberg et al., Mol. Endocrinol. 8:757-70, 1994). In addition, RZRs preferred a T at the -1 position to the core motif; at position -2, all nucleotides were comparable in binding efficiency. RZR receptor transactivation activity closely paralleled DNA binding efficiency. When the two core motif half-sites were present in certain configurations tested, RZRs could bind cooperatively as homodimers. Further, transactivation by RZRs appeared to be constitutive.
The Rev-erba and Rev-erbp orphan nuclear receptors are highly related to each other and to the (ROR)/RZR subfamily of receptors (B. M. Forman et al., Molec. Endocrinol. 8:1253-61, 1994). The Rev-erb receptors bind as monomers to a Rev-erb/ROR.alpha.1 common response element sequence, AATGT-AGGTCA. However, while ROR.alpha.1 constitutively activates transcription through this sequence, both Rev-erb isoforms are inactive. When coexpressed with ROR.alpha.1, the Rev-erb isoforms suppress the transcriptional activity of ROR.alpha.1.
The natural ligands for certain members of the nuclear hormone receptor family have been identified recently. For instance, 9-cis retinoic acid has been identified as the natural ligand for the RXR orphan receptor family (A. A. Levin et al., Nature 355:359-61, 1992; R. A. Heyman et al., Cell 68:397-406, 1992).
Melatonin has been identified as the natural ligand for RZR.beta. receptors (M. Becker-Andre et al., J. Biol. Chem. 269:28531-34, 1994). In the central nervous system, RZR.beta. mRNA is most prominent in pineal gland, thalamus and hypothalamus, while in the periphery only the adrenal gland was reported as positive. A more detailed examination localized RZR.beta. mRNA in the neural retina, the suprachiasmatic nucleau and the superficial gray layer of the superior colliculus. This tissue distribution is consistent with the distribution of binding sites for melatonin. In cells transfected with RZR.beta., fetal calf serum (FCS) increased basal constitutive activity of RZR.beta.. This activation was ameliorated when FCS was treated with charcoal or anti-melatonin antibody plus protein A-Sepharose. A component of FCS was speculated to be involved in post-translational modification of RZR.beta. (i.e., perhaps phosphorylation by protein kinases).
Subsequently, melatonin was reported to bind to RZR.alpha. and its splice variant ROR.alpha.1 (I. Wiesenberg et al., Nucl. Acids Res. 23:327-33, 1995). In contrast to RZR.beta., RZR.alpha./ROR.alpha.1 receptors are expressed in many tissues outside the brain. A synthetic RZR ligand, thiazolidinedione CGP 52608, also binds to RZR.alpha./ROR.alpha.1. However, this synthetic ligand does not bind to the high affinity membrane receptor for melatonin. Further, a class of thiazolidinediones exhibited anti-arthritic effects in vivo that correlated with RZR/ROR.alpha. activation (M. Missbach et al., J. Biol. Chem. 271:13515-22, 1996).
Thiazolidinediones have also been described as high affinity ligands for PPAR.gamma., which functions in adipogenesis (J. M. Lehmann et al., J. Biol. Chem. 270:12953-56, 1995). Thiazolidinedione derivatives are anti-diabetic agents that act as insulin sensitizers. A prostaglandin J.sub.2 (PGJ.sub.2) metabolite, 15-deoxy-.DELTA..sup.12,14 -PGJ.sub.2, binds to PPAR.gamma. also, and promoted differentiation of fibroblasts to adipocytes (S. A. Kliewer et al., Cell 83:813-19, 1996).
Recently, 5-lipoxygenase has been reported to be an RZR/melatonin responding gene (D. Steinhilber et al., J. Biol. Chem. 270:7037-40, 1995). This enzyme is not found in the brain, but instead acts in the periphery, particularly in myeloid cells.
Another ROR/RZR receptor, ROR.gamma., has been reported to be highly expressed in skeletal tissue (T. Hirose et al., Biochem. Biophys. Res. Comm. 205:1976-83, 1994). The amino acid sequence of ROR.gamma. was 50% and 51% identical to those of ROR.alpha./RZR.alpha. and RZR.beta., respectively, with DNA binding domains having 89% identity.
Because orphan nuclear receptors generally regulate important biological processes, identification of novel orphan nuclear receptors furthers an understanding of the complex transcriptional networks in which such receptors function. Identification and characterization of new orphan receptors permits further dissection of the activities of related receptors. A novel orphan receptor provides a means to find the natural ligand for that receptor. By identifying the corresponding ligand, the physiological role of the orphan receptor can be determined.
The present invention provides a novel orphan nuclear receptor for these and other uses that should be apparent to those skilled in the art from the teachings herein.